PROJECT SUMMARY Every part of the brain is composed of dense tangles of heavily-interconnected neurons of many different types, each playing completely different roles in the circuitry. Rabies viral vectors have become indispensable tools for revealing the organization of this otherwise generally indecipherable jumble, because they allow the identification of synaptically-connected networks of neurons within the tissue. They also allow monitoring and manipulation of the activity of the virally-labeled neurons by optogenetics and other techniques, so that the contributions to mental processes of identified components of neural circuitry can be revealed. Despite their usefulness, however, rabies viral vectors have drawbacks, chief among which is their toxicity to the labeled neurons, which prevents their use in longer-term physiological and behavioral experiments that would provide major insights into the biological bases of cognition. In this project, we will develop a new generation of rabies viral vectors and monosynaptic tracing systems based on them that will allow completely nontoxic fluorescent labeling, optical monitoring, and optogenetic manipulation of synaptically-connected neuronal networks.